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Abstract:

The present invention provides a therapeutic agent, for diseases in which
neoplastic proliferation of plasma cells occurs, which is characterized
by containing an anti-human CD48 monoclonal antibody and having cellular
cytotoxicity to cells expressing human CD48; and the monoclonal antibody.
In addition, the present invention provides a method for screening an
active ingredient of a therapeutic agent for diseases in which neoplastic
proliferation of plasma cells occurs, the method including the following
processes: (1) a process of sorting a substance that binds specifically
to human CD48; and (2) a process of sorting a substance having cellular
cytotoxicity.

Claims:

1. An anti-human CD48 monoclonal antibody set forth in any of the
following (a) to (c): (a) a monoclonal antibody that has a heavy chain
variable region comprising an amino acid sequence shown in SEQ ID NO: 1
and a light chain variable region comprising an amino acid sequence shown
in SEQ ID NO: 3, and that binds specifically to human CD48, (b) a
monoclonal antibody that recognizes the same epitope as the monoclonal
antibody described in (a), and (c) a monoclonal antibody that has a heavy
chain variable region comprising an amino acid sequence having an
identity of 90% or higher to the amino acid sequence shown in SEQ ID NO:
1 and a light chain variable region comprising an amino acid sequence
having an identity of 90% or higher to the amino acid sequence shown in
SEQ ID NO: 3, and that binds specifically to human CD48.

2. An anti-human CD48 monoclonal antibody described in any of the
following (d) to (f): (d) a monoclonal antibody that has a heavy chain
variable region comprising an amino acid sequence shown in SEQ ID NO: 1,
a light chain variable region comprising an amino acid sequence shown in
SEQ ID NO: 3, a heavy chain constant region comprising an amino acid
sequence shown in SEQ ID NO: 5, and a light chain constant region
comprising an amino acid sequence shown in SEQ ID NO: 6, and that binds
specifically to human CD48, (e) a monoclonal antibody that recognizes the
same epitope as the monoclonal antibody described in (d), and (f) a
monoclonal antibody that has an amino acid sequence having an identity of
80% or higher to the entire amino acid sequence of the monoclonal
antibody described in (d), that has a heavy chain variable region
comprising an amino acid sequence having an identity of 90% or higher to
the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable
region comprising an amino acid sequence having an identity of 90% or
higher to the amino acid sequence shown in SEQ ID NO: 3, and that binds
specifically to human CD48.

3. The antibody according to claim 1, wherein the antibody is a chimeric
antibody or a humanized antibody.

4. A therapeutic agent for diseases in which neoplastic proliferation of
plasma cells occurs, the therapeutic agent containing an anti-human CD48
monoclonal antibody and having cellular cytotoxicity to cells expressing
human CD48.

5. The therapeutic agent according to claim 4, wherein the monoclonal
antibody has cellular cytotoxicity.

6. The therapeutic agent according to claim 4, wherein the monoclonal
antibody is bound to a substance having cellular cytotoxicity.

7. A therapeutic agent for diseases in which neoplastic proliferation of
plasma cells occurs, the therapeutic agent containing an anti-human CD48
monoclonal antibody and having cellular cytotoxicity to cells expressing
human CD48, wherein the monoclonal antibody is a monoclonal antibody
according to claim 1.

8. A therapeutic method for diseases in which neoplastic proliferation of
plasma cells occurs, the therapeutic method comprising a process of
administrating a therapeutic agent according to claim 4 to a patient
afflicted with a disease in which neoplastic proliferation of plasma
cells occurs.

9. A method for screening an active ingredient for therapy for diseases
in which neoplastic proliferation of plasma cells occurs, the method
comprising the following processes: (1) a process of sorting a substance
that binds specifically to human CD48; and (2) a process of sorting a
substance having cellular cytotoxicity.

10. A method for identifying neoplastic plasma cells, the method
comprising a process of causing a monoclonal antibody to human CD48 to
act on a sample taken from a patient afflicted with a disease in which
neoplastic proliferation of plasma cells occurs.

11. The method according to claim 10, wherein the monoclonal antibody is
an anti-human CD48 monoclonal antibody set forth in any of the following
(a) to (c): (a) a monoclonal antibody that has a heavy chain variable
region comprising an amino acid sequence shown in SEQ ID NO: 1 and a
light chain variable region comprising an amino acid sequence shown in
SEQ ID NO: 3, and that binds specifically to human CD48 (b) a monoclonal
antibody that recognizes the same epitope as the monoclonal antibody
described in (a), and (c) a monoclonal antibody that has a heavy chain
variable region comprising an amino acid sequence having an identity of
90% or higher to the amino acid sequence shown in SEQ ID NO: 1 and a
light chain variable region comprising an amino acid sequence having an
identity of 90% or higher to the amino acid sequence shown in SEQ ID NO:
3, and that binds specifically to human CD48.

12. A reagent or kit for monitoring diseases in which neoplastic
proliferation of plasma cells occurs, the reagent or kit comprising an
anti-human CD48 monoclonal antibody.

13. The reagent or kit according to claim 12, wherein the monoclonal
antibody is an anti-human CD48 monoclonal antibody set forth in any of
the following (a) to (c): (a) a monoclonal antibody that has a heavy
chain variable region comprising an amino acid sequence shown in SEQ ID
NO: 1 and a light chain variable region comprising an amino acid sequence
shown in SEQ ID NO: 3, and that binds specifically to human CD48 (b) a
monoclonal antibody that recognizes the same epitope as the monoclonal
antibody described in (a), and (c) a monoclonal antibody that has a heavy
chain variable region comprising an amino acid sequence having an
identity of 90% or higher to the amino acid sequence shown in SEQ ID NO:
1 and a light chain variable region comprising an amino acid sequence
having an identity of 90% or higher to the amino acid sequence shown in
SEQ ID NO: 3, and that binds specifically to human CD48.

14. The antibody according to claim 2, wherein the antibody is a chimeric
antibody or a humanized antibody.

15. A therapeutic agent for diseases in which neoplastic proliferation of
plasma cells occurs, the therapeutic agent containing an anti-human CD48
monoclonal antibody and having cellular cytotoxicity to cells expressing
human CD48, wherein the monoclonal antibody is a monoclonal antibody
according to claim 2.

16. A therapeutic agent for diseases in which neoplastic proliferation of
plasma cells occurs, the therapeutic agent containing an anti-human CD48
monoclonal antibody and having cellular cytotoxicity to cells expressing
human CD48, wherein the monoclonal antibody is a monoclonal antibody
according to claim 3.

17. A therapeutic method for diseases in which neoplastic proliferation
of plasma cells occurs, the therapeutic method comprising a process of
administrating a therapeutic agent according to claim 5 to a patient
afflicted with a disease in which neoplastic proliferation of plasma
cells occurs.

18. A therapeutic method for diseases in which neoplastic proliferation
of plasma cells occurs, the therapeutic method comprising a process of
administrating a therapeutic agent according to claim 6 to a patient
afflicted with a disease in which neoplastic proliferation of plasma
cells occurs.

19. A therapeutic method for diseases in which neoplastic proliferation
of plasma cells occurs, the therapeutic method comprising a process of
administrating a therapeutic agent according to claim 7 to a patient
afflicted with a disease in which neoplastic proliferation of plasma
cells occurs.

20. A therapeutic method for diseases in which neoplastic proliferation
of plasma cells occurs, the therapeutic method comprising a process of
administrating a therapeutic agent according to claim 15 to a patient
afflicted with a disease in which neoplastic proliferation of plasma
cells occurs.

Description:

TECHNICAL FIELD

[0001] The present invention provides new finding concerning therapy for
diseases in which neoplastic proliferation of plasma cells occurs. More
particularly, the present invention provides finding concerning a novel
target molecule effective for therapy for diseases in which neoplastic
proliferation of plasma cells occurs. The present invention relates to a
novel therapeutic agent and a novel therapeutic method, based on such
finding, for diseases in which neoplastic proliferation of plasma cells
occurs. In addition, the present invention relates to a method for
screening an active ingredient for therapy for diseases in which
neoplastic proliferation of plasma cells occurs. Further, the present
invention relates to a reagent or kit for monitoring diseases in which
neoplastic proliferation of plasma cells occurs.

BACKGROUND ART

[0002] Diseases in which neoplastic proliferation of plasma cells occurs
are diseases in which plasma cells present in bone marrow become
cancerous and proliferate into monoclonal cells. In the case of multiple
myeloma that is a typical example of such diseases, abnormal plasma cells
(myeloma cells) spread in bone marrow all over the body, and proliferate
all over bone marrow in the whole body. The proliferation of the abnormal
plasma cells (myeloma cells) causes various symptoms such as destruction
of bone. The myeloma cells produce M protein, which is an abnormal
immunoglobulin, and the M protein concentration rises in blood, whereby
blood becomes viscous. M protein does not function as a proper antibody
to recognize foreign substances, such as a pathogen that invades the
body, and thus also causes decrease of immunity. These factors affect
many organs and various signs occur. Typical signs are pain and damage of
bone, hypercalcemia, renal damage, renal failure, anemia, etc.

[0003] Multiple myeloma occupies about 1% of all the cancers and occupies
a little more than 10% of all the hematological malignant tumors. Thus,
an effective therapeutic agent therefor is called for. At present,
chemotherapies, such as combined use of merphalan and prednisone and use
of thalidomide, and hematopoietic stem cell transplantation are mainly
performed as therapy for multiple myeloma. However, in most cases,
myeloma cells acquire resistance to these chemotherapeutic agents soon.
Thus, in the existing therapeutic approach, the average survival time
after development of symptoms is about three to five years, and the
prognoses of myeloma patients are actually severe. Since these
therapeutic agents do not act specifically on only target tumor cells,
they exhibit toxicity also to normal cells and there is a problem that
serious side effects are produced as a result.

[0004] One of the reasons why diseases in which neoplastic proliferation
of plasma cells occurs, such as multiple myeloma, are very intractable is
thought to be that myeloma stem cells, which are precursors of myeloma
plasma cells, are not eliminated by therapy (Non-Patent Literature 1 and
Non-Patent Literature 2). Since myeloma stem cells are present in a
CD19+ cell fraction, therapy for multiple myeloma using an antibody
(rituximab) to CD20 highly-expressed in the same pattern as CD19 has been
attempted, but there has been no report that a sufficient therapeutic
efficacy is obtained (Non-Patent Literature 3). In addition, development
of therapy for multiple myeloma using an antibody has been attempted. For
example, it is thought that IL-6 is a major proliferator for multiple
myeloma cells (Non-Patent Literature 4 and Non-Patent Literature 5), and
development of a therapeutic agent for multiple myeloma using a
neutralizing antibody to IL-6 or an IL-6 receptor was attempted for the
purpose of preventing an IL-6 signal transduction system. However,
although proliferation inhibition of myeloma cells was observed in
patients with plasma cell leukemia, tumors recurred and clinical efficacy
has not been obtained (Non-Patent Literature 6 and Non-Patent Literature
7). Further, there have been reports that some antigen molecules (e.g.,
CD19 (Non-Patent Literature 8), CD20 (Non-Patent Literature 9), CD38
(Non-Patent Literature 10), CD54 (Non-Patent Literature 11), CD138
(Non-Patent Literature 12), Muc-1 (Non-Patent Literature 13), etc.) can
be effective targets in antibody therapy, but a practical therapeutic
agent has not been developed.

[0018] An object of the present invention is to provide a novel
therapeutic agent effective for therapy for diseases in which neoplastic
proliferation of plasma cells occurs. In addition, an object of the
present invention is to provide a method for screening an active
ingredient of a therapeutic agent for diseases in which neoplastic
proliferation of plasma cells occurs. Further, an object of the present
invention is to provide novel findings concerning therapy for diseases in
which neoplastic proliferation of plasma cells occurs.

Solution to Problem

[0019] Under the circumstances described above, in order to achieve
radical therapy for diseases in which neoplastic proliferation of plasma
cells occurs, the inventors of the present invention conducted thorough
research concerning a therapeutic method for multiple myeloma as a
typical example. As a result, the inventors found that as shown in
Examples 3 and 4 described below, human CD48 is a molecule that is
consistently expressed on the cell surfaces of myeloma stem cells and
myeloma precursor cells and is not expressed in hematopoietic stem cells.
In addition, the inventors found that human CD48 continues to be highly
expressed not only in myeloma stem cells and myeloma precursor cells but
also in mature myeloma plasma cells. Based on the findings, the inventors
of the present invention further conducted research, prepared a
monoclonal antibody that has cellular cytotoxicity and specifically
recognizes human CD48, and confirmed that by administering the antibody
into animals in which myeloma cells are transplanted proliferation of the
myeloma cells is inhibited. On the basis of theses results, the inventors
confirmed that radical therapy for diseases in which neoplastic
proliferation of plasma cells occurs, such as multiple myeloma, is
possible by targeting cells expressing human CD48 and killing the cells
or inhibiting proliferation of the cells. The present invention is
completed on the basis of the finding described above.

[0020] In other words, the present invention includes inventions of the
following aspects.

I. Therapeutic Agent for Diseases in which Neoplastic Proliferation of
Plasma Cells Occurs

[0021] (I-1) A therapeutic agent for diseases in which neoplastic
proliferation of plasma cells occurs, the therapeutic agent containing a
monoclonal antibody to human CD48 and having cellular cytotoxicity to
cells expressing human CD48.

[0023] (I-3) The therapeutic agent according to (I-1), wherein the
anti-human CD48 monoclonal antibody is bound to a substance having
cellular cytotoxicity.

[0024] (I-4) A therapeutic agent for diseases in which neoplastic
proliferation of plasma cells occurs, the therapeutic agent containing,
as an active ingredient, a product in which a substance having cellular
cytotoxicity is bound to the anti-human CD48 monoclonal antibody.

[0025] (I-5) A therapeutic agent for diseases in which neoplastic
proliferation of plasma cells occurs, the therapeutic agent containing a
substance having cellular cytotoxicity and an anti-human CD48 monoclonal
antibody as its carrier.

[0026] (I-6) The therapeutic agent according to any one of (I-3) to (I-5),
wherein the substance having cellular cytotoxicity is a substance having
an anticancer effect.

[0027] (I-7) The therapeutic agent according to any one of (I-1) to (I-6),
wherein the anti-human CD48 monoclonal antibody is any one of antibodies
of the following (a) to (c):

[0029] (b) a monoclonal antibody that recognizes the same epitope as the
monoclonal antibody described in (a), and

[0030] (c) a monoclonal antibody that has a heavy chain variable region
comprising an amino acid sequence having an identity of 90% or higher to
the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable
region comprising an amino acid sequence having an identity of 90% or
higher to the amino acid sequence shown in SEQ ID NO: 3, and that binds
specifically to human CD48.

[0031] (I-8) The therapeutic agent according to any one of (I-1) to (I-6),
wherein the anti-human CD48 monoclonal antibody is any one of antibodies
of the following (d) to (f):

[0033] (e) a monoclonal antibody that recognizes the same epitope as the
monoclonal antibody described in (d), and

[0034] (f) a monoclonal antibody comprising an amino acid sequence having
an identity of 80% or higher to an entire amino acid sequence of the
monoclonal antibody described in (d), having a heavy chain variable
region comprising an amino acid sequence having an identity of 90% or
higher to the amino acid sequence shown in SEQ ID NO: 1 and a light chain
variable region comprising an amino acid sequence having an identity of
90% or higher to the amino acid sequence shown in SEQ ID NO: 3, and
having a specific binding property to human CD48.

[0035] (I-9) The therapeutic agent according to any one of (I-1) to (I-8),
wherein a disease in which neoplastic proliferation of plasma cells
occurs is multiple myeloma.

II. Method for Screening an Active Ingredient of a Therapeutic Agent for
Diseases in which Neoplastic Proliferation of Plasma Cells Occurs

[0036] (II-1) A method for screening an active ingredient of a therapeutic
agent for diseases in which neoplastic proliferation of plasma cells
occurs, the method comprising the following processes:

[0037] (1) a process of sorting a substance that binds specifically to
human CD48; and

[0038] (2) a process of sorting a substance having cellular cytotoxicity.

III. Method for Identifying Neoplastic Plasma Cells

[0039] (III-1) A method for identifying neoplastic plasma cells, the
method comprising a process of causing a monoclonal antibody to human
CD48 (anti-human CD48 monoclonal antibody) to act on a sample taken from
a patient afflicted with a disease in which neoplastic proliferation of
plasma cells occurs.

[0040] (III-2) The method according to (III-1), wherein the anti-human
CD48 monoclonal antibody is any one of antibodies of the following (a) to
(c):

[0041] (a) a monoclonal antibody that has a heavy chain variable region
comprising an amino acid sequence shown in SEQ ID NO: 1 and a light chain
variable region comprising an amino acid sequence shown in SEQ ID NO: 3,
and that has a specific binding property to human CD48,

[0042] (b) a monoclonal antibody that recognizes the same epitope as the
monoclonal antibody described in (a), and

[0043] (c) a monoclonal antibody that has a heavy chain variable region
comprising an amino acid sequence having an identity of 90% or higher to
the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable
region comprising an amino acid sequence having an identity of 90% or
higher to the amino acid sequence shown in SEQ ID NO: 3, and that binds
specifically to human CD48.

[0044] (III-3) The method according to (III-1) or (III-2), wherein the
anti-human CD48 monoclonal antibody is any one of antibodies of the
following (d) to (f):

[0046] (e) a monoclonal antibody that recognizes the same epitope as the
monoclonal antibody described in (d), and

[0047] (f) a monoclonal antibody that has an amino acid sequence having an
identity of 80% or higher to an entire amino acid sequence of the
monoclonal antibody described in (d), that has a heavy chain variable
region comprising an amino acid sequence having an identity of 90% or
higher to the amino acid sequence shown in SEQ ID NO: 1 and a light chain
variable region comprising an amino acid sequence having an identity of
90% or higher to the amino acid sequence shown in SEQ ID NO: 3, and that
binds specifically to human CD48.

IV. Reagent or Kit for Monitoring Progression of or Therapeutic Effect
for Diseases in which Neoplastic Proliferation of Plasma Cells Occurs

[0048] (IV-1) A reagent or kit for monitoring diseases in which neoplastic
proliferation of plasma cells occurs, the reagent or kit comprising a
monoclonal antibody to human CD48 (an anti-human CD48 monoclonal
antibody).

[0049] (IV-2) The reagent or kit according to (III-1), wherein the
anti-human CD48 monoclonal antibody is any one of antibodies of the
following (a) to (c):

[0051] (b) a monoclonal antibody that recognizes the same epitope as the
monoclonal antibody described in (a), and

[0052] (c) a monoclonal antibody that has a heavy chain variable region
comprising an amino acid sequence having an identity of 90% or higher to
the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable
region comprising an amino acid sequence having an identity of 90% or
higher to the amino acid sequence shown in SEQ ID NO: 3, and that binds
specifically to human CD48.

[0053] (IV-3) The reagent or kit according to (IV-1) or (IV-2), wherein
the anti-human CD48 monoclonal antibody is any one of antibodies of the
following (d) to (f):

[0055] (e) a monoclonal antibody that recognizes the same epitope as the
monoclonal antibody described in (d), and

[0056] (f) a monoclonal antibody that has an amino acid sequence having an
identity of 80% or higher to an entire amino acid sequence of the
monoclonal antibody described in (d), that has a heavy chain variable
region comprising an amino acid sequence having an identity of 90% or
higher to the amino acid sequence shown in SEQ ID NO: 1 and a light chain
variable region comprising an amino acid sequence having an identity of
90% or higher to the amino acid sequence shown in SEQ ID NO: 3, and that
binds specifically to human CD48.

[0058] (V-2) An anti-human CD48 monoclonal antibody that recognizes the
same epitope as the monoclonal antibody according to the (V-1)

[0059] (V-3) An anti-human CD48 monoclonal antibody that has a heavy chain
variable region comprising an amino acid sequence having an identity of
90% or higher to an amino acid sequence shown in SEQ ID NO: 1 and a light
chain variable region comprising an amino acid sequence having an
identity of 90% or higher to an amino acid sequence shown in SEQ ID NO:
3, and that binds specifically to human CD48.

[0061] (V-5) An anti-human CD48 monoclonal antibody that recognizes the
same epitope as the monoclonal antibody described in (V-4).

[0062] (V-6) A monoclonal antibody that has an amino acid sequence having
an identity of 80% or higher to an entire amino acid sequence of the
monoclonal antibody described in (V-4), that has a heavy chain variable
region comprising an amino acid sequence having an identity of 90% or
higher to an amino acid sequence shown in SEQ ID NO: 1 and a light chain
variable region comprising an amino acid sequence having an identity of
90% or higher to an amino acid sequence shown in SEQ ID NO: 3, and that
binds specifically to human CD48.

VI. Therapeutic Method for Diseases in which Neoplastic Proliferation of
Plasma Cells Occurs

[0063] (VI-1) A therapeutic method for diseases in which neoplastic
proliferation of plasma cells occurs, the method comprising a process of
administrating a therapeutic agent according to any one of (I-1) to (I-9)
to a patient afflicted with a disease in which neoplastic proliferation
of plasma cells occurs.

[0064] (VI-2) The therapeutic method according to (VI-1), wherein a
disease in which neoplastic proliferation of plasma cells occurs is
multiple myeloma.

VII. Use for Therapy for Diseases in which Neoplastic Proliferation of
Plasma Cells Occurs

[0065] (VII-1) A monoclonal antibody to human CD48 (an anti-human CD48
monoclonal antibody) used for therapy for diseases in which neoplastic
proliferation of plasma cells occurs.

[0067] (VII-3) The anti-human CD48 monoclonal antibody according to
(VII-1), wherein the anti-human CD48 monoclonal antibody recognizes the
same epitope as the monoclonal antibody described in the (VII-2).

[0068] (VII-4) The anti-human CD48 monoclonal antibody according to
(VII-1), wherein the anti-human CD48 monoclonal antibody is a monoclonal
antibody having a heavy chain variable region that has an amino acid
sequence having an identity of 90% or higher to an amino acid sequence
shown in SEQ ID NO: 1 and a light chain variable region comprising an
amino acid sequence having an identity of 90% or higher to an amino acid
sequence shown in SEQ ID NO: 3, and that binds specifically to human
CD48.

[0070] (VII-6) The anti-human CD48 monoclonal antibody according to
(VII-1), wherein the anti-human CD48 monoclonal antibody is a monoclonal
antibody that recognizes the same epitope as the monoclonal antibody
described in (VII-5).

[0071] (VII-7) The anti-human CD48 monoclonal antibody according to
(VII-1), wherein the anti-human CD48 monoclonal antibody is a monoclonal
antibody that has an amino acid sequence having an identity of 80% or
higher to an entire amino acid sequence of the monoclonal antibody
described in (VII-5), that has a heavy chain variable region comprising
an amino acid sequence having an identity of 90% or higher to an amino
acid sequence shown in SEQ ID NO: 1 and a light chain variable region
comprising an amino acid sequence having an identity of 90% or higher to
an amino acid sequence shown in SEQ ID NO: 3, and that binds specifically
to human CD48.

[0072] (VII-8) The anti-human CD48 monoclonal antibody according to any of
(VII-1) to (VII-7), wherein the anti-human CD48 monoclonal antibody has
cellular cytotoxicity.

[0073] (VII-9) Use of an anti-human monoclonal antibody, for producing a
therapeutic agent for diseases in which neoplastic proliferation of
plasma cells occurs.

Advantageous Effects of Invention

[0074] According to the therapeutic agent and the therapeutic method of
the present invention, by targeting CD48, not only mature myeloma cells
but also a series of undifferentiated myeloma stem cells and myeloma
precursor cells that have a high possibility of differentiating into
myeloma cells in the future can be targeted. Then, by killing and/or
inhibiting proliferation of these target cells, myeloma stem cells and
myeloma precursor cells can be blocked from differentiating into myeloma
cells. Further, since CD48 is expressed in mature myeloma plasma cells as
well, according to the therapeutic agent and the therapeutic method of
the present invention, the mature myeloma plasma cells is killed and/or
proliferation thereof is inhibited. Thus, the therapeutic agent of the
present invention can be used effectively for therapy, particularly
radical therapy, for diseases in which neoplastic proliferation of plasma
cells occurs, such as multiple myeloma. In addition, the therapeutic
agent of the present invention has low affinity to hematopoietic stem
cells, and thus a safety problem caused by hematopoietic stem cells being
attacked is alleviated.

[0075] According to the screening method of the present invention, use of
a binding property to human CD48 and cellular cytotoxicity as indicators
makes it possible to easily and efficiently obtain an active ingredient
for therapy for diseases in which neoplastic proliferation of plasma
cells occurs. In addition, the method of the present invention allows
neoplastic plasma cells to be identified easily and more accurately, and
makes it possible to monitor diseases in which neoplastic proliferation
of plasma cells occurs.

BRIEF DESCRIPTION OF DRAWINGS

[0076] FIG. 1 shows the morphological features of cells contained in a
myeloma stem cell fraction, a myeloma precursor cell fraction, and a
mature myeloma cell fraction, by May-Giemsa staining.

[0085] FIG. 10 shows the volume changes of tumor masses formed by a
myeloma cell line subcutaneously transplanted in Rag2-/-cγ-/- mice
(an anti-CD48 antibody administration group and a control IgG
administration group).

[0086] FIG. 11 shows the sizes of the tumor masses in the
Rag2-/-cγ-/- mice (the anti-CD48 antibody administration group and
the control IgG administration group) on Day 12, wherein the arrow
indicates the width of the tumor.

[0087] FIG. 12 shows changes of the chimerisms of myeloma cells in the
Rag2-/-cγ-/- mice (the anti-CD48 antibody administration group and
the control IgG administration group) before and after the administration
of an anti-CD48 antibody or mouse IgG.

[0088] FIG. 13 shows a result of comparison of CD48 expression level in
each of bone marrow cell fractions derived from a healthy subject.

[0089] FIG. 14 shows the numbers of various kinds of colony forming cells
obtained by cultivating CD34 positive hematopoietic precursor cells
derived from a healthy subject in the presence of the anti-CD48 antibody
(1B4) or mouse IgG and a complement.

[0090] FIG. 15 shows a result of CD48-CD38 co-staining flow cytometry
analysis for identifying myeloma cells in a myeloma patient.

[0091] In the present specification, a myeloma stem cell and a myeloma
precursor cell refer to cells that are at a stage prior to
differentiation into mature myeloma plasma cells (neoplastic plasma
cells) and that have a property of differentiating into myeloma plasma
cells later. Myeloma stem cells and myeloma precursor cells can be
classified according to the stages of their differentiation. FIG. 1 shows
pictures of myeloma stem cells, myeloma precursor cells, and mature
myeloma plasma cells that are stained with May-Giemsa. Bone marrow B
cells that have become myeloma stem cells turn into mature myeloma plasma
cells through myeloma precursor cells later.

[0092] A "myeloma stem cell (CD19+ myeloma stem cell)" is
characterized by expression of CD19, which is a surface antigen molecule.
Thus, in the present specification, a myeloma stem cell may be described
as "CD19+ cell".

[0093] A "myeloma precursor cell (CD19-CD38++CD138- myeloma
precursor cell)" is a precursor cell that is differentiated from a
CD19+ myeloma stem cell and that is at a stage immediately prior to
differentiation into a myeloma plasma cell. CD38 is highly expressed
therein, but the "myeloma precursor cell" is characterized by
non-expression of CD138, which is a specific marker for mature plasma
cells. Meanwhile, no expression of CD19 is observed. Thus, the myeloma
precursor cell may be described as "CD19-CD38++CD138cell".

[0094] In general, the "myeloma plasma cell" is also referred to as
myeloma cell, and is a cell that produces M protein, which is an abnormal
immunoglobulin. In the myeloma plasma cell, in addition to CD38 being
highly expressed, CD138 is expressed. Meanwhile, no expression of CD19 is
observed. Thus, the myeloma plasma cell may be described as
"CD19-CD38++CD138+ cell". In the present specification,
the myeloma stem cell, the myeloma precursor cell, and the myeloma plasma
cell also refer to a tumor stem cell, a tumor precursor cell, and a
neoplastic plasma cell, respectively, in diseases in which neoplastic
proliferation of plasma cells occurs, other than multiple myeloma.

[0095] A "hematopoietic stem cell" is a cell that can differentiate into
any hematopoietic cell. The hematopoietic stem cell is characterized by
expression of CD34. Thus, in the present specification, the hematopoietic
stem cell may be described as "CD34+ cell".

[0096] In the present invention, diseases in which neoplastic
proliferation of plasma cells occurs are diseases that are characterized
by neoplastic proliferation of abnormal plasma cells and increase of
abnormal proteins secreted from them. Particular examples of neoplastic
proliferation of plasma cells include multiple myeloma, plasmacytic
leukemia, plasmocytoma, H chain diseases, and systemic AL type
amyloidosis. A disease to be treated by a therapeutic agent of the
present invention is not particularly limited as long as it is a disease
in which neoplastic proliferation of plasma cells occurs, but is
preferably multiple myeloma.

I. Therapeutic Agent and Therapeutic Method for Diseases in which
Neoplastic Proliferation of Plasma Cells Occurs

[0097] The therapeutic agent of the present invention for diseases in
which neoplastic proliferation of plasma cells occurs contains an
anti-human CD 48 monoclonal antibody and has cellular cytotoxicity to
cells expressing human CD48.

I-I. Monoclonal Antibody to Human CD48

[0098] The monoclonal antibody to human CD48 (hereinafter, may be referred
to as "anti-human CD48 monoclonal antibody") is a monoclonal antibody
that binds specifically to human CD48. As shown in Examples 3 and 4
described below, human CD48 is consistently expressed on the cell
surfaces of myeloma stem cells, myeloma precursor cells, and myeloma
plasma cells, but is not expressed at all or is slightly expressed in
hematopoietic stem cells. Thus, by targeting human CD48, myeloma stem
cells, myeloma precursor cells, and myeloma plasma cells can be targeted
without targeting hematopoietic stem cells which supply normal
lymphocytes and the like. The anti-human CD48 monoclonal antibody is an
antibody that binds specifically to human CD48, and thus can specifically
recognize and bind to myeloma stem cells, myeloma precursor cells, and
myeloma plasma cells. Therefore, the anti-human CD48 monoclonal antibody
is an antibody suitable for targeting cells to be treated in therapy,
preferably radical therapy, for diseases in which neoplastic
proliferation of plasma cells occurs, such as multiple myeloma. In other
words, by combining the anti-human CD48 monoclonal antibody with cellular
cytotoxicity, the activity can be caused to act specifically on myeloma
stem cells, myeloma precursor cells, and myeloma plasma cells.

[0099] The anti-human CD48 monoclonal antibody intended by the present
invention include various antibodies, as long as they have a property of
recognizing and binding to myeloma stem cells, myeloma precursor cells,
and myeloma plasma cells due to affinity to human CD48 as described
above. For example, the anti-human CD48 monoclonal antibody may be a
modified antibody such as a humanized antibody, or may be its fragment
(e.g., Fab, Fab', F(ab')2, scFv, etc.).

[0100] A preferable anti-human CD48 monoclonal antibody is, for example, a
monoclonal antibody produced in Example 4 described below (hereinafter,
may be referred to as "1B4 antibody"), has an amino acid sequence shown
in SEQ ID NO: 1 as the amino acid sequence of a heavy chain variable
region, has an amino acid sequence shown in SEQ ID NO: 3 as the amino
acid sequence of a light chain variable region, has an amino acid
sequence shown in SEQ ID NO: 5 as the amino acid sequence of a heavy
chain constant region, and has an amino acid sequence shown in SEQ ID NO:
6 as the amino acid sequence of a light chain constant region. The 1B4
antibody can be produced on the basis of its sequence information using a
genetic engineering technique or a chemical peptide synthesis method
known in the technical field.

[0101] Other preferable monoclonal antibodies are antibodies that
recognize the same epitope as the 1B4 monoclonal antibody, and, in
particular, monoclonal antibodies that can bind to the same epitope.
Whether or not an antibody recognizes the same epitope as another
antibody can be confirmed by competition of these antibodies to epitope.
Competition between antibodies can be assessed by a competitive binding
assay, and examples of its means include an enzyme-linked immunosorbent
assay (ELISA), a fluorescent-energy-transfer measuring method (FRET) and
fluorometric microvolume assay technology (FMAT (registered trademark)).
The amount of the antibody binding to an antigen indirectly correlates
with the binding capacity of a candidate competitive antibody (a test
antibody) that competes for binding to the same epitope. In other words,
as the amount and affinity of the test antibody to the same epitope
increases, the amount of the antibody binding to the antigen decreases
and the amount of the test antibody binding to the antigen increases.
More particularly, the appropriately-labeled antibody and an antibody
that is to be assessed are simultaneously added to the antigen, and the
binding antibody is detected using the label. The amount of the antibody
binding to the antigen can easily be measured when the antibody is
previously labeled. This labeling is not particularly limited, but a
labeling method suitable for the technique is selected. Particular
examples of the labeling method include fluorescence labeling,
radiolabeling, and enzyme labeling.

[0102] For example, the fluorescence-labeled antibody and the unlabeled
antibody or the test antibody are simultaneously added to beads obtained
by solidifying human CD48, and the labeled antibody is detected by
fluorometric microvolume assay technology.

[0103] The "antibody that recognizes the same epitope" herein is an
antibody that is a test antibody that can decrease the binding amount of
the labeled antibody by at least 50% with a concentration that is higher,
by normally 100 times, preferably 80 times, more preferably 50 times,
even more preferably 30 times, and much more preferably 10 times, than a
concentration (IC50) of the unlabeled antibody with which the
binding amount of the labeled antibody is decreased by 50% due to binding
of the unlabeled antibody.

[0104] Examples of such monoclonal antibodies include the following
antibodies (A) and (B).

[0105] (A) Antibody: an antibody comprising an amino acid sequence that is
an amino acid sequence of the 1B4 antibody in which one or a plurality of
amino acids are substituted, deleted, inserted, and/or added.

[0106] (B) Antibody: an antibody comprising an amino acid sequence having
an identity of 80% or higher to the entire amino acid sequence of the 1B4
antibody.

[0107] In the above antibody (A), the plurality of amino acids are, for
example, 2 to 30 amino acids, preferably 2 to 15 amino acids, more
preferably 2 to 10 amino acids, even more preferably 2 to 5 amino acids,
and much more preferably 2 or 3 amino acids. The positions at which the
amino acids are substituted, deleted, inserted, and/or added are not
limited as long as the antibody specifically recognizes the same epitope
as the 1B4 antibody, but the positions are preferably regions other than
CDR1 to 3 in the heavy chain and the light chain shown in FIGS. 6 and 7
and more preferably constant regions. The substitution, deletion,
insertion, or addition of one or a plurality of amino acids can be
performed according to a known method described in Molecular Cloning, A
Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press,
1989) or the like.

[0108] The identity of the amino acid sequence of the above antibody (B)
is preferably equal to or higher than 85%, more preferably equal to or
higher than 90%, even more preferably equal to or higher than 95%, and
particularly preferably equal to or higher than 98%. In a preferred
embodiment, the above antibody (B) has a heavy chain variable region
having an identity of 90% or higher, preferably 95% or higher, more
preferably 97% or higher, and particularly preferably 98% or higher, to
the amino acid sequence shown in SEQ ID NO: 1, and has a light chain
variable region having an identity of 90% or higher, preferably 95% or
higher, more preferably 97% or higher, and particularly preferably 98% or
higher, to the amino acid sequence of SEQ ID NO: 3. In still another
preferred embodiment, the above antibody (B) has the same amino acid
sequence as the 1B4 antibody in the CDR1 to 3 of the heavy chain and the
light chain.

[0109] An identity of amino acids can be calculated using an analysis tool
(e.g., software such as FASTA, BLAST, PSI-BLAST, and SSEARCH) that is
commercially available or can be used through the Internet. For example,
main initial conditions generally used for BLAST searching are as
follows. Particularly, in Advanced BLAST 2.1, blastp is used as a
program, an Expect value is set to 10, all Filters are set to OFF,
BLOSUM62 is used as Matrix, Gap existence cost, Per residue gap cost, and
Lambda ratio are set to 11, 1, 0.85 (defaults), respectively, other
various parameters are also set to defaults, and searching is performed,
whereby the value (%) of an identity of an amino acid sequence can be
calculated.

[0110] The anti-human CD48 monoclonal antibody may be an antibody
belonging to any immunoglobulin class and subclass, but is preferably an
antibody belonging to human immunoglobulin class and subclass. The class
and subclass are preferably immunoglobulin G (IgG) and more preferably
human IgG1.

[0111] The anti-human CD48 monoclonal antibody can be produced according
to a known method described in Molecular Cloning, A Laboratory Manual,
Second Edition (Cold Spring Harbor Laboratory Press, 1989) or the like. A
particular production method will be described below. In addition, some
anti-human CD48 monoclonal antibodies are commercially available, and
they can also be selected/prepared as appropriate and used.

[0112] In light of reducing antigenicity to human, the anti-human CD48
monoclonal antibody is preferably a humanized antibody. The humanized
antibody is a chimeric antibody that is obtained by substituting, with
the amino acid sequence of a human immunoglobulin, a portion of a
non-human animal antibody other than the variable region (or the super
variable region) and that has reduced antigenicity to human while
maintaining its affinity to human CD48. The humanized monoclonal antibody
can be produced according to a known method.

Method for Producing Anti-Human CD48 Monoclonal Antibody

[0113] The anti-human CD48 monoclonal antibody can be produced by
immunizing an animal with human CD48. Hereinafter, a method for producing
a monoclonal antibody to human CD48 will be described more particularly.

(1) Obtaining of Human CD48

[0114] First, a DNA fragment containing a cDNA coding for human CD48 is
inserted into an appropriate expression vector to create a recombinant
vector. This vector is introduced into a host cell suitable for the
expression vector, to obtain a transformant. The DNA coding for human
CD48 is known (e.g., database NCBI Genbank, accession number NM 001778.2)
and is also commercially available. As the host cell, arbitrary cells,
such as Escherichia coli, yeast, insect cells, and animal cells, can be
used as long as they can express human CD48. As the expression vector,
arbitrary expression vectors can be used as long as they have an
appropriate promoter that can transcribe the DNA coding for CD48, in the
host cell. The introduction of the recombinant vector into the host can
be performed by a method selected as appropriate from known methods such
as an electroporation method, a calcium phosphate method, and a
lipofection method.

[0115] The obtained transformant is cultivated in an appropriate medium to
express human CD48, and the human CD48 can be collected and obtained. As
an immunogen for producing the antibody to human CD48, other than human
CD48, a transformed cell expressing human CD48 may be used without any
change, or may be isolated/purified as necessary and used.

[0116] Further, human CD48 can be produced using a chemical synthesis
method such as a tBoc method (t-butyloxy carbonyl method) or an Fmoc
method (fluorenyl methyloxy carbonyl method).

(2) Immunization with Human CD48

[0117] Animals are immunized with the human CD48 obtained as described
above as an antigen, and antibody-producing cells are taken from spleen
or lymph node. The kind of the immunized animals is not particularly
limited, and, for example, can be selected as appropriate from mouse,
rat, hamster, rabbit, goat, monkey, pig, horse, etc. The immunization can
be performed by subcutaneously, intravenously, or intraperitoneally
administrating the human CD48 antigen to the animals. An appropriate
adjuvant may be added with the antigen to boost immunoresponsiveness of
the immunized animals to the antigen. Normally, after the first
administration of the antigen, the immunization is performed 2 to 5 times
at intervals of 4 days to 2 weeks. A blood sample is collected from
fundus venous plexus on Days 3 to 7 after each administration of the
antigen, and its blood serum is used to measure reactivity with human
CD48. A test animal exhibiting a sufficient antibody titer can be used as
a supply source of antibody-producing cells.

[0118] The anti-human CD48 monoclonal antibody can be obtained by fusing
antibody-producing cells and myeloma cells to create hybridomas, and
cultivating the hybridomas. The antibody-producing cells can be obtained
from the spleen of an animal that is confirmed to exhibit a sufficient
antibody titer. The origin of the myeloma cells is not particularly
limited, but the same kind of animal as the test animal is preferably
used. For example, when a mouse is used as an immunized animal and
antibody-producing cells are obtained, a mouse-derived cell line (e.g., a
myeloma cell line derived from BALB/c mouse) is preferably used.

(3) Cell Fusion

[0119] Cell fusion can be performed using a known method such as a method
using polyethylene glycol (a PEG method), a method using Sendai virus, or
a method using an electrofusion device. When the PEG method is used,
antibody-producing cells and bone marrow cells are mixed in an
appropriate medium or buffer containing about 30 to 60% of PEG (average
molecular weight 1000 to 6000), such that antibody-producing cell:myeloma
cell=5 to 10:1, and are reacted with each other under the conditions of
about 25 to 37° C. and pH6 to 8 for about 30 seconds to 3 minutes,
whereby the antibody-producing cells and the bone marrow cells can be
fused.

[0120] Selection of hybridoma can be performed by cultivating the fused
cells in a selection medium. The selection medium is not particularly
limited as long as it is a medium in which the parent cell line is killed
and only the fused cells can proliferate. Normally, a
hypoxanthine-aminopterin-thymidine medium (HAT medium) is used. After the
cell fusion reaction ends, the cells are washed, and the PEG solution is
removed. Then, in the selection medium cultivation, cultivation is
performed while repeating medium replacement every 2 or 3 days, whereby
selection of hybridoma is possible.

[0121] At the end, for the selected hybridoma, the affinity to human CD48
is measured by the following method, and an anti-human CD48 monoclonal
antibody can be obtained.

(4) Measurement of Affinity to Human CD48

[0122] The affinity of the monoclonal antibody and its fragment to CD48
can be measured by any method known in the technical field. For example,
the affinity can be measured by the following method. First, two types of
cells, cells in which human CD48 is expressed and cells in which human
CD48 is not expressed, are prepared. These two types of cells are the
same except for presence/absence of expression of human CD48. Next, a
fluorescence-labeled test antibody or its fragment is provided to each
cell, and presence/absence of binding between the cells and the antibody
or its fragment is measured using flow cytometry. An antibody that binds
only to cells expressing human CD48 has specific affinity to human CD48,
and an antibody that does not bind only to cells expressing human CD48 is
an antibody having no or low specific affinity to human CD48. Further,
the degree of the affinity of the monoclonal antibody to CD48 can be
measured by the strength of a fluorescent signal detected by flow
cytometry.

[0123] Other than the method using flow cytometry, an immunoassay can also
be used to measure the affinity. In this case, a microtiter plate is
coated with purified human CD48, and a test antibody or its fragment is
added as a first antibody to each well to cause a reaction. Next, an
antibody (second antibody) that can recognize the first antibody and that
is labeled with an enzyme, a fluorescent substance, a luminous substance,
a radioactive substance, or biotin is added to react with the first
antibody. Then, the affinity of the test antibody or its fragment to
human CD48 can be measured using the label of the second antibody as an
indicator.

(5) In Light of Reducing Antigenicity to Human, the Human CD48 Monoclonal
Antibody is Preferably a Humanized Antibody.

[0124] The humanized antibody (humanized anti-human CD48 monoclonal
antibody) can be created according to any method known in the technical
field. For example, first, a hybridoma producing a monoclonal antibody to
human CD48 is created using non-human animal cells. Next, a DNA fragment
coding for the amino acid sequences of the heavy and light chain variable
regions (or super variable regions) of a non-human animal antibody that
is produced from the hybridoma is obtained. This fragment is bound to a
DNA coding for the amino acid sequence of a portion of a human-derived
antibody other than the variable region (or super variable region), to
create a DNA coding for a humanized antibody. At the end, the DNA is
expressed in animal cells using an appropriate animal cell expression
vector, to obtain the humanized antibody.

[0125] More particularly, a cDNA fragment coding for a heavy chain
variable region comprising the amino acid sequence shown in SEQ ID NO: 1
and a cDNA fragment coding for a light chain variable region comprising
the amino acid sequence shown in SEQ ID NO: 3 are inserted into a
chimeric antibody production vector (e.g., an expression vector described
in Reff M E et. al. Blood 83 435-445, 1994), and the vector is
transfected into CHO cells to express the cDNAs, whereby an anti-human
CD48 chimerized antibody can be produced. A particular example of the
amino acid sequences of the H chain constant region and the L chain
constant region of a specific human IgH gamma 1 antibody that can be used
for such humanization is shown below.

A humanized antibody having lower antigenicity can be produced by
substituting, with human-derived sequences, all the portions other than
the super variable regions (CDR1, 2, and 3) in addition to the constant
regions.

[0128] As the anti-human CD48 monoclonal antibody, its fragment may be
used as long as it has affinity to human CD48. Examples of the fragment
of the anti-human CD48 monoclonal antibody include Fab, Fab',
F(ab')2, and scFv.

[0129] The Fab fragment is a fragment in which the L chain and the H chain
are bound to each other via a disulfide bond, among fragments obtained by
treating the antibody molecule with a protease papain. Thus, the Fab
fragment of the anti-human CD48 monoclonal antibody can be obtained by
treating the anti-human CD48 monoclonal antibody with papain, or can be
obtained by inserting a DNA coding for the Fab fragment of the anti-human
CD48 monoclonal antibody into an arbitrary expression vector and
expressing the DNA in an appropriate host cell.

[0130] The F(ab')2 fragment is a fragment that is obtained by
treating the antibody molecule with a protease pepsin and in which two
Fab fragments are bound to each other via a disulfide bond in a hinge
region, and maintains affinity to the antigen. Thus, the F(ab')2
fragment of the anti-human CD48 monoclonal antibody can be obtained by
treating the anti-human CD48 monoclonal antibody with pepsin.

[0131] The Fab' fragment is a fragment that is obtained by cutting the
disulfide bond in the hinge region of the above F(ab')2 fragment and
in which a full-length light chain and a heavy chain from the N terminal
to the hinge region are bound to each other via a disulfide bond, and
maintains affinity to the antigen. Thus, the Fab' fragment of the
anti-human CD48 monoclonal antibody can be obtained by treating the above
F(ab')2 fragment, for example, with a reducing agent such as
dithiothreitol to cut the disulfide bond in the hinge region. In
addition, the Fab' fragment of the anti-human CD48 monoclonal antibody
can also be obtained by inserting a DNA coding for the Fab' fragment into
an arbitrary expression vector and expressing the DNA in an appropriate
host cell.

[0132] The scFv fragment is a fragment in which the variable regions of
the light chain and the heavy chain are bound to each other using a
peptide linker and that maintains affinity to the antigen. Thus, the
scFab' fragment of the anti-human CD48 monoclonal antibody can be
obtained by constructing a DNA coding for the scFv fragment from a cDNA
coding for the anti-human CD48 monoclonal antibody such that the length
of a linker is preferably equal to or less than 8 amino acids, inserting
the DNA into an arbitrary expression vector, and expressing the DNA in an
appropriate host cell.

[0133] The anti-human CD48 monoclonal antibody obtained as described above
can recognize myeloma stem cells, myeloma precursor cells, and myeloma
plasma cells. Here, if the anti-human CD48 monoclonal antibody can have
cellular cytotoxicity in addition to this recognition ability, the
anti-human CD48 monoclonal antibody can kill and/or inhibit proliferation
of myeloma stem cells, myeloma precursor cells, and myeloma plasma cells,
and can be used effectively as an active ingredient of a therapeutic
agent for diseases in which neoplastic proliferation of plasma cells
occurs, such as myeloma.

[0134] Such a combination of cell recognition ability and cellular
cytotoxicity is possible by (1) using an anti-human CD48 monoclonal
antibody itself having cellular cytotoxicity or (2) binding another
substance having cellular cytotoxicity to an anti-human CD48 monoclonal
antibody. Hereinafter, each embodiment will be described.

[0135] In one embodiment, the present invention is a therapeutic agent,
for diseases in which neoplastic proliferation of plasma cells occurs,
which contains, as an active ingredient, a monoclonal antibody that is an
antibody to the human CD48 and that has cellular cytotoxicity
(hereinafter, also referred to as "anti-human CD48 cellular cytotoxicity
monoclonal antibody").

I-II-I. Anti-Human CD48 Cellular Cytotoxicity Monoclonal Antibody

[0136] The anti-human CD48 cellular cytotoxicity monoclonal antibody is an
antibody that is the aforementioned anti-human CD48 monoclonal antibody
and that has cellular cytotoxicity. Here, the "cellular cytotoxicity"
means a property of being able to kill and/or inhibit proliferation of
myeloma stem cells, myeloma precursor cells, and myeloma plasma cells.
Thus, as long as such an effect is provided, its action and mechanism are
not particularly limited. For example, this activity is provided by one
or a combination of two or more of complement-dependent cytotoxicity
(CDC), antibody-dependent cellular cytotoxicity (ADCC), apoptosis
induction, inhibition of a survival signal by blocking of ligand binding,
and the like.

[0137] The anti-human CD48 cellular cytotoxicity monoclonal antibody
intended by the present invention includes various antibodies, as long as
they have cellular cytotoxicity and affinity to CD48. For example, it may
be a modified antibody such as a humanized antibody, or may be its
fragment (e.g., Fab, Fab', F(ab')2, scFv, etc.). A preferable
anti-human CD48 cellular cytotoxicity monoclonal antibody is, for
example, a monoclonal antibody that has the amino acid sequence shown in
SEQ ID NO: 1 as the amino acid sequence of a heavy chain variable region,
the amino acid sequence shown in SEQ ID NO: 3 as the amino acid sequence
of a light chain variable region, the amino acid sequence shown in SEQ ID
NO: 5 as the amino acid sequence of a heavy chain constant region, and
has the amino acid sequence shown in SEQ ID NO: 6 as the amino acid
sequence of a light chain constant region. Other preferable monoclonal
antibodies are antibodies that recognize the same epitope as the 1B4
monoclonal antibody, particularly monoclonal antibodies that can bind to
the same epitope as this antibody, and more particularly the monoclonal
antibodies described in the above I-I.

[0138] Whether or not an antibody has cellular cytotoxicity can be
measured according to a known method. For example, complement-dependent
cytotoxicity or antibody-dependent cellular cytotoxicity can be measured
by the following method.

[0140] Complement-dependent cytotoxicity can be measured according to a
method of Brunner K. T., et al. (Brunner, K. T., et al., Immunology,
1968. 14:181-96). For example, myeloma cells that are target cells are
cultivated in a RPMI1640 medium in which 10% FCS is added, and are
prepared such that the number of cells is 0.5×104 to
1.0×104. An appropriate amount of Na251CrO4 is
added thereto, a reaction is allowed to proceed at 37° C. for one
hour, and the cells are labeled with 51Cr and washed to give target
cells. A test antibody or isotype antibody, which is control, suspended
in a fetal calf serum-added RPMI1640 medium is added to a 96-well plate
such that the final concentration is 0.5 to 50 μg/mL, and then the
target cells and a complement are added, and a reaction is allowed to
proceed for 1.5 hours. The reaction solution is centrifuged, 51Cr
released to the supernatant is measured with a γ-counter. The CDC
activity can be obtained on the basis of the following equation.

CDC activity={([51Cr release from cells used in
experiment]-[voluntary 51Cr release in state where there is no
antibody]/([maximum 51Cr release amount by addition of 1% Triton
X-100]-[voluntary 51Cr release in state where there is no
antibody])}×100

[0142] Antibody-dependent cellular cytotoxicity can be measured according
to a method of Brunner K. T., et al. (Brunner, K. T., et al., Immunology,
1968. 14:181-96). For example, as target cells, the same
51Cr-labeled myeloma-related cells as in the above case of the
measurement of complement-dependent cytotoxicity can be used. As effector
cells, SCID mouse bone marrow cells that are cultivated for six days in
RPMI1640 in which 10% FBS, 10 ng/ml of mouse GM-CSF, and 40 IU/ml of
human IL2 are added, or the like can be used. An antibody or its isotype
antibody that is control is added to a 96-well plate such that the final
concentration is 0.05 to 10 μg/mL, and the target cells
(1.0×104 cells) and the effector cells (5×105
cells) are further added. A reaction is allowed to proceed at 37°
C. for four hours, centrifugation is performed, and then 51Cr
released to the supernatant is measured with a γ-counter. The ADCC
activity can be obtained on the basis of the following equation.

ADCC activity={([51Cr release from cells used in
experiment]-[voluntary 51Cr release in state where there is no
antibody]/([maximum 51Cr release amount by addition of 1% Triton
X-100]-[voluntary 51Cr release in state where there is no
antibody])}×100

[0143] The antibody, to human CD48, having cellular cytotoxicity can be
obtained by producing anti-human CD48 monoclonal antibodies, assessing
presence/absence of cellular cytotoxicity using the above method, and
selecting an antibody having this activity.

[0144] The anti-human CD48 cellular cytotoxicity monoclonal antibody can
bind specifically to myeloma stem cells, myeloma precursor cells, and
myeloma plasma cells and can kill and/or inhibit proliferation of myeloma
stem cells, myeloma precursor cells, and myeloma plasma cells, and thus
is useful as an active ingredient of a therapeutic agent, particularly a
radical therapeutic agent, for diseases in which neoplastic proliferation
of plasma cells occurs, such as multiple myeloma.

[0145] The therapeutic agent of the present invention in the present
embodiment may contain only the anti-human CD48 cellular cytotoxicity
monoclonal antibody as an active ingredient, but may further contain, as
necessary, pharmaceutically acceptable one or more additives, for
example, one or more of a diluent, a preservative, a solubilizer, an
emulsifier, an adjuvant, an antioxidant, an isotonizing agent, an
excipient, and a carrier. In addition, the therapeutic agent may be a
mixture with another antibody or another agent such as an antibiotic.
Suitable carriers include, but are not limited to, a physiological
saline, a phosphate buffered saline, a phosphate buffered saline glucose
solution, and a buffered saline solution. Further, stabilizing agents
such as amino acids, sugars, and surfactants, and inhibitors for
adsorption to surface, which are known in this field, may be contained.
As the form of a formulation, formulations including a lyophilized
formulation (in this case, the formulation can be reconstructed and used
by adding the above buffered solution), a slow release formulation, an
enteric coated formulation, an injection, and drops are selectable
according to aim of therapy and therapy planning.

[0146] As a route of administration of the therapeutic agent of the
present invention, any of oral administration and parenteral
administration (e.g., intraoral, tracheobronchial, rectal, subcutaneous,
intramuscular, and intravenous) may be used, as long as the therapeutic
effect for diseases in which neoplastic proliferation of plasma cells
occurs is provided. Since the active ingredient contains the antibody,
parenteral administration is preferred, and intravenous administration is
further preferred. Thus, a preferable administration form is an
injection. The injection is prepared using a carrier consisting of a salt
solution, a glucose solution, or a mixture thereof, or the like.

[0147] Dosages and the number of times of administration of the
therapeutic agent of the present invention depend on an intended
therapeutic effect, an administration method, therapy duration, age, body
weight, and the like, but the therapeutic agent can be administered to an
adult patient afflicted with multiple myeloma, normally in an amount of
50 μg to 0.5 mg/kg per day.

[0148] I-III. Therapeutic Agent Containing One in which a Substance Having
Cellular Cytotoxicity is Bound to Anti-Human CD48 Monoclonal Antibody

[0149] In one embodiment, the present invention is a therapeutic agent,
for diseases in which neoplastic proliferation of plasma cells occurs,
which contains, as an active ingredient, one in which a substance having
cellular cytotoxicity is bound to an anti-human CD48 monoclonal antibody.

[0150] The active ingredient of the therapeutic agent in the present
embodiment is one in which a substance having cellular cytotoxicity is
bound to an anti-human CD48 monoclonal antibody. As described above, the
anti-human CD48 monoclonal antibody specifically recognize myeloma stem
cells, myeloma precursor cells, and myeloma plasma cells. Thus, by
binding the substance having cellular cytotoxicity to the anti-human CD48
monoclonal antibody, the substance can be transferred to myeloma stem
cells, myeloma precursor cells, and myeloma plasma cells and caused to
act specifically on these cells. In other words, by binding the substance
having cellular cytotoxicity to the anti-human CD48 monoclonal antibody,
the substance can be prevented from nonspecifically acting. Therefore, by
the present invention, it is possible to treat diseases in which
neoplastic proliferation of plasma cells occurs, such as multiple
myeloma, while avoiding side effects caused by nonspecific action of the
substance having cellular cytotoxicity on cells other than the above
cells.

[0151] The anti-human CD48 monoclonal antibody used in the present
embodiment is not particularly limited as long as it has affinity to
human CD48, and includes the aforementioned various monoclonal antibodies
(e.g., a modified antibody such as humanized antibody, and its fragment).
Due to the nature of the invention, the anti-human CD48 monoclonal
antibody does not have to itself have cellular cytotoxicity, but may be a
monoclonal antibody having cellular cytotoxicity.

I-III-I. Substance Having Cellular Cytotoxicity

[0152] The substance having cellular cytotoxicity is a substance having a
property of, when being bound to the anti-human CD48 monoclonal antibody
and transferred to myeloma stem cells, myeloma precursor cells, and
myeloma cells, being able to kill and/or inhibit proliferation of these
cells. Since targeting and transferring to myeloma stem cells, myeloma
precursor cells, and myeloma plasma cells are performed by the antibody,
the substance having cellular cytotoxicity may not be one itself acting
specifically on myeloma stem cells, myeloma precursor cells, and myeloma
cells, as long as it has cellular cytotoxicity. Here, cellular
cytotoxicity means a property of being able to kill and/or inhibit
proliferation of cells. As long as such an effect is provided, its
mechanism is not particularly limited, and an arbitrary substance can be
used, but typical substances having cellular cytotoxicity are compounds
known as anticancer agents. Particular examples include alkylating agents
such as cyclophosphamide hydrate, ifosfamide, thiotepa, busulfan,
merphalan, nimustine hydrochloride, ranimustine, dacarbazine, and
temozolomide; antimetabolites such as methotrexate, pemetrexed sodium
hydrate, fluorouracil, doxifluridine, capecitabine, tegafur, cytarabine,
gemcitabine hydrochloride, fludarabine phosphate, nelarabine, cladribine,
and levofolinate calcium; antibiotics such as doxorubicin hydrochloride,
daunorubicin hydrochloride, pirarubicin, epirubicin hydrochloride,
idarubicin hydrochloride, aclarubicin hydrochloride, amrubicin
hydrochloride, mitoxantrone hydrochloride, mitomycin C, actinomycin D,
bleomycin hydrochloride, peplomycin hydrochloride, zinostatin stimalamer,
and calicheamicin; microtubule inhibitors such as vincristine sulfate,
vinblastine sulfate, vindesine sulfate, and paclitaxel; aromatase
inhibitors such as anastrozole, exemestane, letrozole, and fadrozole
hydrochloride hydrate; platinum agents such as cisplatin, carboplatin,
nedaplatin, and oxaliplatin; topoisomerase inhibitors such as irinotecan
hydrochloride hydrate, nogitecan hydrochloride, etoposide, and
sobuzoxane; adrenocorticosteroids such as prednisolone and dexamethasone;
thalidomide and lenalidomide that is its derivative, and bortezomib that
is a protease inhibitor. Among them, calicheamicin, merphalan,
vincristine sulfate, doxorubicin hydrochloride, prednisolone,
dexamethasone, thalidomide, lenalidomide, and bortezomib are preferred,
and calicheamicin, which has produced good results in binding to an
antibody, is more preferred. Any of the above-exemplified substances
having cellular cytotoxicity are commercially available. As the substance
having cellular cytotoxicity, one or more substances can be selected,
bound to the anti-human CD48 monoclonal antibody, and used.

[0153] Alternatively, a radioisotope such as 90-Ittrium can be bound to
the anti-human CD48 monoclonal antibody and used.

I-III-II. Method for Binding Substance Having Cellular Cytotoxicity and
Antibody

[0154] The substance having cellular cytotoxicity and the anti-human CD48
monoclonal antibody can be bound to each other by any method known in the
technical field, as long as the cellular cytotoxicity of the substance
and the affinity of the antibody to CD48 are not impaired. The substance
and the antibody may be directly bound to each other or may be indirectly
bound to each other via a linker or the like. The binding may be either a
covalent bond or a noncovalent bond (e.g., ionic bond). For example, a
reactive group (e.g., an amino group, a carboxyl group, a hydroxyl group,
etc.) or a coordinating group in the antibody molecule is used to make
the antibody contact with a cellular cytotoxicity substance having a
functional group (in the case of a bacterial toxin or a chemotherapeutic
agent) that can react with the reactive group to form a bond or having an
ionic group (in the case of a radionuclide) that can form a complex with
the coordinating group, whereby the antibody and the cellular
cytotoxicity substance can be bound to each other. In addition, it is
also possible to use biotin and avidin when forming a composite body.
When the cellular cytotoxicity substance is a protein or peptide, a
fusion protein of the antibody and the protein or peptide can be produced
by a genetic engineering technique. In light of maintaining the affinity
of the antibody, for example, the antibody and the substance having
cellular cytotoxicity are preferably bound to each other via an amino
acid present in an Fc fragment.

[0155] By binding the substance having cellular cytotoxicity to the
anti-human CD48 antibody as described above, the active ingredient of the
therapeutic agent of the present invention is obtained. The therapeutic
agent of the present invention may be composed of only the cellular
cytotoxicity substance and the anti-human CD48 antibody, but may contain
one or more pharmaceutically acceptable additives as necessary. As
additives, those described in the above I-II. can be used. In addition,
the therapeutic agent of the present invention may be a mixture with
another antibody or another agent such as an antibiotic. As a carrier and
the like, those described in the above I-II. can be used. As the form of
a formulation, formulations including a lyophilized formulation (in this
case, the formulation can be reconstructed and used by adding the above
buffered solution), a slow release formulation, an enteric coated
formulation, an injection, and drops are selectable according to aim of
therapy and therapy planning.

[0156] As a route of administration of the therapeutic agent of the
present invention, any of oral administration and parenteral
administration (e.g., intraoral, tracheobronchial, rectal, subcutaneous,
intramuscular, and intravenous) may be used, as long as the therapeutic
effect for diseases in which neoplastic proliferation of plasma cells
occurs is provided. Since the active ingredient contains the antibody,
parenteral administration is preferred, and intravenous administration is
further preferred. Thus, a preferable administration form is an
injection. The injection is prepared using a carrier consisting of a salt
solution, a glucose solution, or a mixture thereof, or the like. Dosages
and the number of times of administration of the therapeutic agent of the
present invention depend on an intended therapeutic effect, an
administration method, therapy duration, age, body weight, and the like,
but the therapeutic agent can be administered to an adult patient
afflicted with a disease in which neoplastic proliferation of plasma
cells occurs, for example, in an amount of 1 to 9 mg/m2 body surface
area per day.

II. Method for Screening Active Ingredient of Therapeutic Agent for
Diseases in which Neoplastic Proliferation of Plasma Cells Occurs

[0157] In one embodiment, the present invention is a method, for screening
an active ingredient of a therapeutic agent for diseases in which
neoplastic proliferation of plasma cells occurs, which includes (1) a
process of sorting a substance that binds specifically to human CD48 and
(2) a process of sorting a substance having cellular cytotoxicity. The
substance searched for and obtained by the method of the present
invention can bind specifically to human CD48 and has cellular
cytotoxicity. Therefore, it is expected that when being administered to
patients afflicted with diseases in which neoplastic proliferation of
plasma cells occurs such as multiple myeloma, the substance specifically
recognizes myeloma stem cells, myeloma precursor cells, and myeloma
plasma cells and exerts cytotoxic effects thereon, whereby diseases in
which neoplastic proliferation of plasma cells occurs can be treated.

[0158] The substance to be screened by the present invention is not
particularly limited as long as it is one sorted by the processes (1) and
(2), and is preferably an antibody.

[0159] The process (1) is a process of sorting a substance that
specifically recognizes and binds to human CD48, and can be performed
according to the methods described in (4) in the above "Method for
producing anti-human CD48 monoclonal antibody", but is not limited
thereto.

[0160] Hereinafter, a particular method is exemplified. Two types of
cells, cells in which human CD48 is expressed and cells in which human
CD48 is not expressed, are prepared. These two types of cells are the
same except for presence/absence of expression of human CD48. Next, a
fluorescence-labeled test substance is provided to each cell, and
presence/absence of binding between the cells and the substance is
measured using flow cytometry. A substance that binds only to cells
expressing human CD48 has specific affinity to human CD48, and an
antibody that does not bind only to cells in which human CD48 is
expressed is an antibody having no or low specific affinity to human
CD48. The degree of the affinity of the antibody to CD48 can be measured
by the strength of a fluorescent signal detected by flow cytometry.

[0161] Other than the method using flow cytometry, an immunoassay can also
be used to measure the affinity. In this case, a microtiter plate is
coated with purified human CD48, and a test substance is added to each
well to cause a reaction. Next, an antibody that can recognize the
substance and that is labeled with an enzyme, a fluorescent substance, a
luminous substance, a radioactive substance, or biotin is added to react
with the substance. Then, the affinity of the test substance to human
CD48 can be measured using the label of the antibody as an indicator.

[0162] The test substance is not particularly limited as long as the
affinity to human CD48 can be measured by these methods, but is
preferably an antibody.

[0163] The process (2) is a process of sorting a substance having cellular
cytotoxicity, and the cellular cytotoxicity can be measured using the
methods described in the above I-II-II. and I-II-III. for measuring
complement-dependent cytotoxicity (CDC) and for measuring
antibody-dependent cellular cytotoxicity (ADCC).

[0164] The candidate substances sorted as described above can further be
sorted out as a more practical active ingredient of a therapeutic agent
for multiple myeloma, by further conducting a drug effect test, a
stability test, etc. using sick nonhuman animals with diseases in which
neoplastic proliferation of plasma cells occurs.

III. Method for Identifying Neoplastic Plasma Cells

[0165] In one embodiment, the present invention is a method for
identifying neoplastic plasma cells, including a process of causing an
anti-human CD 48 monoclonal antibody to act on a sample taken from a
patient afflicted with a disease in which neoplastic proliferation of
plasma cells occurs. As described above, the anti-human CD48 monoclonal
antibody is an antibody that binds specifically to human CD48 to
specifically recognize myeloma stem cells, myeloma precursor cells, and
myeloma plasma cells. Thus, the anti-human CD48 monoclonal antibody is
caused to act on the sample containing neoplastic plasma cells, and an
antibody that binds to cells expressing CD48 is detected, whereby it is
possible to identify the neoplastic plasma cells in the sample. Here, the
sample is a sample (e.g., bone marrow, blood, tumor, etc.) that is taken
from a patient afflicted with a disease in which neoplastic proliferation
of plasma cells occurs and that contains neoplastic plasma cells, is
preferably body fluid, and is further preferably blood. In order to
facilitate detection, the anti-human CD48 monoclonal antibody may be
modified with a fluorescent dye or radioisotope. The identification of
myeloma plasma cells may be performed using the anti-human CD48
monoclonal antibody alone or in combination with another antibody (e.g.,
an anti-human CD38 monoclonal antibody). For example, a sample taken from
the bone marrow of a myeloma patient is co-stained with the
fluorescence-labeled anti-human CD48 monoclonal antibody and the
anti-human CD38 monoclonal antibody, and cells in the sample are
separated for CD38 and CD48 by flow cytometry, whereby the myeloma cell
population can easily be identified. When these monoclonal antibodies are
used, the myeloma plasma cells can be identified as cells that have
strong positivity for CD38 and CD48 (e.g., see Example 9).

IV. Reagent or Kit for Monitoring Progression of or Therapeutic Effect
for Diseases in which Neoplastic Proliferation of Plasma Cells Occurs

[0166] In one embodiment, the present invention relates to a reagent or
kit, for monitoring progression of or a therapeutic effect for diseases
in which neoplastic proliferation of plasma cells occurs, including a
monoclonal antibody to human CD48. As described above, the anti-human
CD48 monoclonal antibody can specifically recognize myeloma stem cells,
myeloma precursor cells, and myeloma plasma cells, and hence these cells
can be identified. Thus, for a patient afflicted with a disease in which
neoplastic proliferation of plasma cells occurs, for example, the
concentrations (numbers) of myeloma stem cells, myeloma precursor cells,
and myeloma plasma cells in blood can be measured using the anti-human
CD48 monoclonal antibody, to monitor the progression of or the
therapeutic effect for the disease. The monitoring reagent of the present
invention may include only the anti-human CD48 monoclonal antibody, and
may additionally include an arbitrary component required for monitoring,
as necessary. The monitoring kit of the present invention may include, in
addition to the anti-human CD48 monoclonal antibody, another component
(e.g., another antibody, a buffer, a fluorescent dye, etc.), an
instrument, a manual, and the like. When monitoring is performed
according to the present invention, it can be used for determination of
therapy planning.

EXAMPLES

Test Method: Flow Cytometry and Sorting

[0167] In the following examples, flow cytometry sorting used for sorting
cells were performed as follows. Bone marrow monocytes taken from the
iliac bone of a myeloma patient from which informed consent was obtained
were suspended in an ACK solution (150 mM NH4Cl and 10 mM
KHCO3) and allowed to stand for 3 minutes at 4° C. to remove
red blood cells. After washing with a PBS (Phosphate-buffered saline) in
which 2% fetal bovine serum was added, in order to prevent nonspecific
binding of an antibody, blocking was performed in a PBS containing 10%
human AB type serum, for 20 minutes at 4° C. Then, each antibody
(see the following) labeled with a fluorescent dye was added to perform
staining for 30 minutes at 4° C., and washing was performed with a
PBS. Then, the bone marrow monocytes were suspended in a PBS containing 1
μg/ml of propidium iodide (PI) and subjected to flow cytometry
analysis. Analysis and cell sorting were performed using a FACS Aria cell
sorter (manufactured by Becton Dickinson Immunocytometry System).

[0169] For radical therapy for multiple myeloma, it is important to target
myeloma stem cells and myeloma precursor cells that are at a stage prior
to differentiation into myeloma plasma cells. Meanwhile, it is also
important to exclude, from targets, hematopoietic stem cells required for
producing normal B cells and plasma cells. In order to target myeloma
stem cells and myeloma precursor cells while excluding hematopoietic stem
cells from targets, it is desired to find a molecule that is not
expressed in hematopoietic stem cells but is expressed in common on the
cell surfaces of myeloma stem cells and myeloma precursor cells, and to
use the molecule as an indicator for target cells. Thus, in order to find
such a molecule, the following screening was performed.

First Screening

[0170] First, genes coding for molecules expressed in myeloma stem cells
and myeloma precursor cells were identified using the following three
methods (A) to (C).

[0171] Myeloma precursor cells (CD19-CD38++CD138- myeloma
precursor cells) were obtained from a multiple myeloma patient, and genes
coding for cell surface proteins were identified from among genes
expressed in these cells.

[0172] First, myeloma precursor cells were separated from bone marrow
cells derived from the multiple myeloma patient, by flow cytometry cell
sorting. Total RNA was taken from the cells using a Trizol reagent
(Invitrogen, Carlsbad, Calif.). Next, cDNA was created from all the RNAs
using a PCR cDNA synthesis kit (SMART: Clontech, Palo Alto, Calif.) and
amplified by PCR to obtain a cDNA library. The cDNA of the library was
cut by a restriction enzyme RsaI, and then a BstXI adaptor was bound
thereto. Then, electrophoresis was performed on 1% agarose gel, cDNA
having a size of 0.5 kb to 2.0 kb was cut out from the gel, purified, and
then inserted into a pMX-SST vector (provided by Professor Toshio
Kitamura of the Institute of Medical Science, the University of Tokyo).
The SST-REX library created as described above was introduced into BaF3
cells according to a signal sequencing trap method described in a report
of Kitamura et al. (Kojima, T. and T. Kitamura, A signal sequence trap
based on a constitutively active cytokine receptor. Nat Biotechnol, 1999.
17(5): p. 487-90), and then screened to comprehensively separate cDNAs
included in the cDNA library and coding for cell surface proteins. Gene
analysis was conducted on the separated cDNAs to identify gene names from
gene sequences. The identified genes are shown in the following Table 1
as genes expressed in myeloma precursor cells.

(B) Second Method: Identification of Genes Highly Expressed in Common in
Myeloma Stem Cells and Myeloma Precursor Cells Using Gene Chip

[0173] Myeloma stem cells (CD19+ cells) and myeloma precursor cells
(CD19-CD38++CD138- cells) were obtained from a multiple
myeloma patient different from the patient from which the cells were
obtained in the above first method, and genes expressed in these cells
were identified.

[0174] Myeloma stem cells (CD19+ cells) and myeloma precursor cells
(CD19-CD38++CD138- cells) were separated from the bone
marrow cells derived from the other myeloma patient, by flow cytometry
sorting (see FIG. 2). Next, total RNA was taken from each of the
separated cell fractions using a Micro RNeasy kit (manufactured by
Qiagen). After cDNA was created from 20 ng of the total RNA using a
GeneChip Two-Cycle cDNA Synthesis Kit (manufactured by Affymetrix), a
1st cycle cRNA was created using a MEGAscript T7 Kit (manufactured
by Ambion) and further a Biotinylated cRNA was created using an IVT
Labeling Kit (manufactured by Affymetrix). After fragmentation was
performed, 11.25 μg of cRNA was hybridized to GeneChip Human Genome
U133 Plus 2.0 Array for 16 hours at 45° C. After the GeneChips
were washed with Affymetrix Fluidics Station 450 and stained, the
GeneChips were scanned using GeneChip Scanner 3000 7G. The result was
obtained using Microarray Suite version 5.0 (MAS5.0), and global scaling
was used as a standardization method. In this manner, molecules highly
expressed in both myeloma stem cells and myeloma precursor cells are
selected from among the identified genes. The selected genes are shown in
Table 2.

(C) Third Method: Searching for Genes Coding for Candidate Molecules on
the Basis of Literature Information

[0175] Genes coding for proteins that can be targets for antibody therapy
were selected from the molecules reported to be expressed in myeloma
cells in a literature. In other words, genes that are not widely
expressed in many organs and that are unclear to be expressed in a CD34
positive hematopoietic stem cell fraction in the released gene expression
database Gene Card (http://www.genecards.org/) were selected from the
cell surface molecules reported to be expressed in myeloma cells in a
literature (e.g., Claudio, J. O., et al., Blood, 2002. 100:2175-86.)
searchable in PubMED (http://www.ncbi.nlm.nih.gov/pubmed/). The selected
genes are shown in Table 3.

[0176] Genes that were actually highly expressed in myeloma stem cells,
myeloma precursor cell, and myeloma plasma cells that were derived from a
plurality of patients were screened from among the candidate genes
obtained by the above first screening. First, molecules (e.g.,
Niemann-Pick disease, type C2 (NPC2) and CD9 molecule) expected to be
ubiquitously expressed in multiple organs from searching of the gene
expression database were excluded from the candidate genes shown in the
above Tables 1 to 3. A PCR primer was produced for each of the remaining
genes. Meanwhile, myeloma stem cells (CD19+ cells) and myeloma
precursor cells (CD19-CD38++CD138- cells) were taken from
three different myeloma patients, and cDNA was created similarly as in
the first method (A). Quantitive PCR was performed using the primer
created for each of the obtained candidate molecules. Quantitive RT-PCR
was performed by an SYBR Green method using ABI 7700 real-time PCR
machine (manufactured by Applied Biosystems). The expression level of
each gene was standardized by the expression level of β-actin. When
molecules having a low possibility of being expressed in a myeloma stem
cell fraction (the difference in Ct value from β-actin is equal to
or higher than 10) were excluded and then molecules observed to have the
same expression level in a myeloma precursor cell fraction as in a
myeloma stem cell fraction were selected, four molecules, CD48 and MMSC2
to MMSC4, were obtained (FIG. 3).

[0177] From the above results, it is confirmed that the CD48 and MMSC2 to
MMSC4 molecules are expressed in common in the myeloma stem cell fraction
and the myeloma precursor cell fraction, and the possibility is suggested
that they can be ideal target molecules.

[0178] Presence/absence of expression of CD48 and MMSC2 to MMSC4 on the
cell surfaces of myeloma stem cells, myeloma precursor cells, myeloma
plasma cells, and hematopoietic stem cells was measured using a
commercially available anti-CD48 antibody (eBioscience) and antibodies to
MMSC2 to MMSC4.

[0179] Bone marrow cells derived from a multiple myeloma patient were
stained with APC-conjugated CD34 (manufactured by BD Pharmingen),
Cy7APC-conjugated CD19 (manufactured by BD pharmingen), Cy7PE-conjugated
CD38 (manufactured by eBioscoiences), PE-conjugated CD138 (manufactured
by BD pharmingen), Biotin-conjugated CD3 (manufactured by BD pharmingen),
Biotin-conjugated CD14 (manufactured by eBiosciences), Cy5PE-conjugated
CD235 (manufactured by Biolegend), and FITC-conjugated CD48 (manufactured
by eBiosciences) (or FITC-conjugated MMSC2 to MMSC4), and washed. Then,
second staining was performed with Cy5PE-conjugated streptoavidin
(manufactured by eBiosciences). As an Isotype control, a sample in which
FITC-conjugated mouse IgG was added instead of FITC-conjugated CD48 and
MMSC2 to MMSC4 was prepared at the same time. These samples were analyzed
using flow cytometry to measure expression distributions of the CD48
molecule and the MMSC2 to MMSC4 molecules in protein level in a
CD19+ myeloma stem cell fraction, a CD19-CD38++CD138myeloma precursor cell fraction, CD138+ mature myeloma plasma cell
fraction, and a CD34+ hematopoietic stem/precursor cell fraction.

[0180] Screening was performed using specimens from myeloma patients of
three cases. An example of the results is shown in FIG. 4. In each
histogram shown in FIG. 4, the Y axis indicates the number of cells, and
the X axis indicates the expression intensity of CD48 or MMSC2 to MMSC4.
From the results shown in FIG. 4, it is confirmed that CD48 is expressed
at a high level in any of the CD19+ myeloma stem cell fraction, the
CD19CD38++CD138- myeloma precursor cell fraction, and the
CD138+ mature myeloma plasma cells, and has a low expression level
in the CD34+ hematopoietic stem/precursor cell fraction. On the
other hand, it is confirmed that the MMSC2 molecule is expressed at a
relatively high level also in the CD34+ hematopoietic stem/precursor
cell fraction. It is confirmed that the MMSC3 molecule and the MMSC4
molecule are not expressed or are expressed at a relatively low level in
the CD19+ myeloma stem cell fraction and the
CD19-CD38++CD138- myeloma precursor cell fraction. As a
result, it is seen that only CD48 is expressed on the cell surfaces of
all of the myeloma stem cells, the myeloma precursor cells, and the
myeloma plasma cells, and has a sufficiently low expression level in the
hematopoietic cells. Thus, it is strongly suggested that the CD48
molecule can be an ideal target molecule indicating cells to be targeted
for therapy, in radical therapy for multiple myeloma. On the other hand,
for MMSC2, since expression in the hematopoietic stem cells is observed,
it is suggested that MMSC2 is not ideal as an indicator indicating target
cells for therapy for multiple myeloma. In addition, for MMSC3 and MMSC4,
since the expression levels on the cell surfaces of the myeloma stem
cells and/or the myeloma precursor cells are low, it is suggested that
MMSC3 and MMSC4 are not ideal as indicators indicating target cells for
therapy for multiple myeloma.

Example 3

[0181] Confirmation of Expression Distribution of CD48 Using Cells Derived
from a Plurality of Patients

[0182] In order to confirm whether the expression distribution of CD48
confirmed in Example 2 is the same as in cells derived from other
patients, bone marrow cells were taken from a plurality of patients, and
expression of CD48 in each cell fraction was measured similarly as in
Example 2. The results of analysis of the bone marrow specimens from
myeloma patients of 10 cases are shown in FIG. 5. From the results shown
in FIG. 5, it becomes clear that the anti-CD48 antibody is bound to the
cell fractions derived from the other patients, in the same pattern as in
the results in Example 3. In other words, it is confirmed that the CD48
molecule is expressed at a high level in any of the myeloma stem cell
fraction and the myeloma precursor cell fraction and has a low expression
level in the hematopoietic stem cell fraction and the hematopoietic
precursor cell fraction.

[0183] As described above, from the results in Examples 2 and 3, it is
confirmed that CD48 is highly expressed on the cell surfaces of the
myeloma stem cells, the myeloma precursor cells, and the myeloma plasma
cells derived from multiple myeloma patients and is not expressed at all
or is expressed at a very low expression level in the hematopoietic stem
cells. Thus, since use of CD48 as an indicator makes it possible to
target a series of cells from the CD19+ myeloma stem cell fraction
to the CD19-CD38++CD138- myeloma precursor cell fraction
and to exclude hematopoietic stem cells from targets, it is strongly
suggested that CD48 is a molecule that becomes an ideal indicator for
radical therapy for multiple myeloma.

Example 4

Creation of Monoclonal Antibody to CD48

[0184] In order to examine whether the CD48 molecule itself is appropriate
as a target for radical therapy for multiple myeloma, a monoclonal
antibody to CD48 was created. First, a human CD48 cDNA (FLJ clone,
manufactured by Toyobo Co., Ltd.) was inserted into an MSCV-ires-GFP
vector, and the vector was introduced into BaF3 cells using retrovirus to
create human CD48-expressing mouse cells. The cells were immunized in
Foot pad of a Balb/c mouse four times, then, the lymph node was taken
out, and cell fusion was performed with mouse myeloma cells SP2/0 to
produce hybridomas. The fused cells were cultivated in an HAT medium.
Hybridomas selected on the basis of presence/absence of proliferation
were cultivated on a microtiter plate, and binding to BaF3 cells in which
CD48 was expressed was observed for the supernatant by flow cytometry to
perform screening, to obtain CD48 antibody producing hybridomas. Through
such a single cell fusion, hybridomas of 4 clones producing the
anti-human CD48 monoclonal antibody were obtained. Among them, it was
confirmed that two clones (1B4 and 2E2) are IgG2a subclass. Confirmation
of the subclass was performed using an Isotyping kit (Roche).

[0185] Further, the base sequences and the amino acid sequences of the
variable regions of antibody molecules produced by the hybridomas 1B4 and
2E2 were determined. The determination of the sequences was performed
according to an already-reported method of Coloma et al. (Coloma M J et
al. Journal of Immunological Methods 152, 89-104, 1992). In other words,
cDNA fragments of the H chain and K chain variable regions were amplified
by a PCR reaction with, as a template, cDNA produced from RNA derived
from each hybridoma, and the base sequences were decoded. The decoded
amino acid sequence (SEQ ID NO: 1) and base sequence (SEQ ID NO: 2) of
the H chain variable region and super variable regions (CDR1 to 3) are
shown in FIG. 6. The decoded amino acid sequence (SEQ ID NO: 3) and base
sequence (SEQ ID NO: 4) of the L chain (K chain) variable regions and
super variable regions (CDR1 to 3) are shown in FIG. 7. In addition,
since the sequence obtained from 1B4 completely coincides with the
sequence derived from 2E2, it becomes clear that these two clones produce
antibodies having exactly the same sequence.

[0186] Presence/absence of cellular cytotoxicity in vitro of the
anti-human CD48 monoclonal antibody created in Example 4 was checked. In
the test, for the monoclonal antibody produced by 1B4 that was confirmed
to belong to IgG2a subclass, presence/absence of complement-dependent
cytotoxicity (CDC) was checked using a chromium release method. A baby
rabbit complement (Cedarene) was used as a complement. As myeloma cells,
myeloma cell lines OPM2 and U266 were used. The OPM2 cell line and the
U266 cell line highly express the CD48 molecule as shown in FIG. 8. Each
myeloma cell line was labeled with 51Cr for two hours and washed
three times. The labeled cells (1×104 cells) were cultivated
in 96-well U-bottomed plates (1×104 cells) in 160 μL of
RPMI1640+fetal bovine serum in which the anti-human CD48 monoclonal
antibody or an isotype control (10 μg/ml in final) and 25% baby rabbit
complement were added. After the cultivation under the conditions of
37° C. and 5% CO2 for 90 minutes, 51Cr released to the
supernatant was counted. Specific cellular cytotoxicity was calculated as
follows.

CDC activity={([51Cr release from cells used in
experiment]-[voluntary 51Cr release in state where there is no
antibody]/([maximum 51Cr release amount by addition of 1% Triton
X-100]-[voluntary 51Cr release in state where there is no
antibody])}×100

[0187] The results of the measurement are shown in FIG. 9. As shown in
FIG. 9, it is confirmed that the 1B4 monoclonal antibody clearly has
cellular cytotoxicity to the myeloma cell lines OPM2 and U266. The
results demonstrate that the 1B4 monoclonal antibody and the antibody
that recognizes the same epitope as the 1B4 monoclonal antibody can be
active ingredients for therapy for multiple myeloma and that checking of
the therapeutic effect in vivo for multiple myeloma using these
monoclonal antibodies makes it possible to check efficacy of treating
multiple myeloma with, as a target, cells expressing CD48.

[0188] The therapeutic effect in vivo for multiple myeloma was checked
using the anti-human CD48 monoclonal antibody confirmed to have cellular
cytotoxicity in Example 5.

[0189] Myeloma cell line OPM2 cells (1×107 cells) were
subcutaneously transplanted into Rag2-/-cy-/- mice irradiated
with radiation of 2 Gy. When the tumor volume exceeded 10 mm3 (10
days after the transplantation of the tumor), the mice were divided into
a CD48 antibody administration group and a control IgG administration
group, and 10 mg/kg of the anti-human CD48 monoclonal antibody (1B4) or
control IgG was administered three times a week (every second days).
Measurement of the tumor volume was performed three times a week (every
second days), and the volume was represented by the following approximate
value: long diameter×short diameter×height/2. The time when
the volume of the tumor mass formed by the transplanted myeloma cell line
OPM2 exceeded 10 mm3 is set to Day 0, and the change of the tumor
volume from that day is shown in FIG. 10. In addition, the sizes of the
tumors in the control (IgG administration) mouse and the 1B4 antibody
administration mouse on Day 12 are shown in FIG. 11. The arrow indicates
the width of the tumor.

[0190] As shown in FIGS. 10 and 11, while the myeloma cells exponentially
proliferated when control IgG was administered, proliferation of the
myeloma cells was almost completely inhibited when the anti-human CD48
monoclonal antibody (1B4) having cellular cytotoxicity was administered.

[0191] In order to check the effect for myeloma cells in a more
physiological environment, the effect of antibody administration for
myeloma cells transplanted in bone marrow was examined. After
3×105 myeloma cell line OPM2 cells were transplanted into the
bone marrows of Rag2-/-cy-/- mice irradiated with radiation of
2 Gy, bone marrow aspiration was performed on Day 10, and the chimerism
of human myeloma cells was analyzed on the basis of the frequency of
hCD38-expressing cells. In addition, 5 mg/kg of the CD48 antibody or
mouse IgG as control was intravenously administered on Day 11, Day 13,
and Day 15. Then, in order to examine the effect of the antibody, the
chimerism of human myeloma cells in bone marrow was analyzed again on Day
16.

[0192] While increase in chimerism of myeloma cells was markedly observed
in any of the mice of the control IgG antibody administration group, the
chimerism of myeloma cells decreased for the CD48 antibody (FIG. 12).
From these experimental results, it becomes clear that the anti-human
CD48 monoclonal antibody (1B4) and the antibody that recognizes the same
epitope as the anti-human CD48 monoclonal antibody have very high
cellular cytotoxicity to myeloma cells expressing CD48. By targeting
CD48, not only myeloma plasma cells but also myeloma stem cells and
myeloma precursor cells can be targeted, and thus the possibility is
strongly suggested that the anti-human CD48 monoclonal antibody is
effective for radical therapy for multiple myeloma. Further, since
specifically killing cells in which human CD48 is expressed, with human
CD48 as an indicator, is effective for radical therapy for multiple
myeloma, it is suggested that a combination of a substance (e.g., a
monoclonal antibody) that specifically recognizes human CD48 and another
substance having cellular cytotoxicity becomes a therapeutic agent
effective for radical therapy for multiple myeloma.

[0193] As shown in FIG. 5, expression of CD48 is slightly observed in
hematopoietic stem cells (CD34+CD38-) and CD34+CD38hematopoietic precursor cells. Also for bone marrow cells derived from a
healthy subject, when the expression level of CD48 was checked similarly
as in Example 3, expression of CD48 was confirmed at a very low level in
CD34+ cells that are fractions of hematopoietic stem cells and
hematopoietic precursor cells (FIG. 13). Thus, it was examined whether
hematopoietic stem cells and hematopoietic precursor cells are subjected
to the cytotoxic effects by the anti-CD48 antibody as a result of
expression of CD48 at such a low level.

[0194] Normal CD34+ cells purified with CD34MACS beads (Miltenyi
Biotec) were caused to react with the anti-human CD48 monoclonal antibody
(1B4) and a complement by the same method as for the measurement of CDC
activity, and then cultivated in a methylcellulose medium (Methocult
H4334 (Stem Cell Technologies)), and the numbers of various colony
forming cells were counted after 14 days. As a result, even with the
cocultivation with the anti-CD48 antibody and the complement, the number
of colonies formed from the normal hematopoietic precursor cells did not
change at all (FIG. 14). The result demonstrates that the hematopoietic
precursor cells are not subjected to cellular injury caused by the
anti-CD48 antibody, and suggests that the anti-CD48 antibody is safe as a
pharmaceutical agent.

[0195] Due to the above, targeting target cells in therapy for multiple
myeloma using the anti-human CD48 monoclonal antibody is thought to be
effective also in terms of safety.

Example 9

[0196] Identification of Myeloma Plasma Cells with Expression Level of
CD48 as Marker

[0197] As is obvious from FIGS. 4 and 5, the expression level of CD48 is
very high in the myeloma precursor cells and the myeloma plasma cells. It
is novel finding that CD48 has strong positivity for CD38-strong-positive
myeloma cells. Using this, for myeloma patient bone marrow, CD48 and CD38
are co-stained and then analyzed by flow cytometry, whereby it is very
easy to identify the myeloma cell population. One example of the analysis
method is shown in FIG. 15. Use of CD48 as a marker together with CD38,
which is in general used as a marker for myeloma cells, as in FIG. 15
allows the myeloma cell population to be identified very easily.
CD38-strong-positive cells are normally identified as myeloma cells, but
a level at which strong positivity is determined is arbitrary. Meanwhile,
addition of CD48 as a marker allows the myeloma cell population to be
regarded as a cell population having uniform and strong positivity for
CD38 and CD48. The frequency, in bone marrow, of the myeloma cell
population identified thus can be measured to recognize the degree of
progression of myeloma.